Protein tyrosine phosphatase mutations in cancers

ABSTRACT

Tyrosine phosphorylation, regulated by protein tyrosine phosphatases (PTPs) and kinases (PTKs), is important in signaling pathways underlying tumorigenesis. A mutational analysis of the tyrosine phosphatase gene superfamily in human cancers identified 83 somatic mutations in six PTPs (PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, PTPN14), affecting 26% of colorectal cancers and a smaller fraction of lung, breast and gastric cancers. Fifteen mutations were nonsense, frameshift or splice site alterations predicted to result in truncated proteins lacking phosphatase activity. Five missense mutations in the most commonly altered PTP (PTPRT) were biochemically examined and found to reduce phosphatase activity. Expression of wild-type but not a mutant PTPRT in human cancer cells inhibited cell growth. These observations suggest that the tyrosine phosphatase genes are tumor suppressor genes, regulating cellular pathways that may be amenable to therapeutic intervention.

This invention was made under contracts (CA43460 and CA63934) with anagency (National Institutes of Health) of the United States Government.The United States Government therefore retains certain rights in theinvention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of cancer. In particular, itrelates to diagnosis, prognosis, treatment, drug discovery, targetdiscovery, clinical testing for cancer.

BACKGROUND OF THE INVENTION

Phosphorylation of tyrosine residues is a central feature of mostcellular signaling pathways, including those affecting growth,differentiation, cell cycle regulation, apoptosis and invasion (1, 2).This phosphorylation is coordinately controlled by protein tyrosinekinases (PTKs) and phosphatases (PTPs). Although a variety of PTK geneshave been directly linked to tumorigenesis through somatic activatingmutations (3-6) only a few PTP genes have been implicated in cancer(7-10). Moreover, it is not known how many or how frequently members ofthe PTP gene family are altered in any particular cancer type.

The PTP gene superfamily is composed of three main families: (i) theclassical PTPs, including the receptor PTPs (RPTPs) and the non-receptorPTPs (NRPTPs); (ii) the dual specificity phosphatases (DSPs), which candephosphorylate serine and threonine in addition to tyrosine residues;and (iii) the low molecular weight phosphatases (LMPs) (1).

There is a continuing need in the art to identify new therapeutictargets, identify new drugs, improve diagnosis, prognosis, and therapyof cancers..

SUMMARY OF THE INVENTION

One aspect of the invention is a method for identifying mutationsinvolved in cancer. Nucleotide sequence differences are determined in ahuman nucleotide sequence between matched pairs of cancer cells andnormal cells. Each matched pair of cells is isolated from a singleindividual. The human nucleotide sequence encodes a protein tyrosinephosphatase selected from the group consisting of: PTPRF, PTPRG, PTPRT,PTPN3, PTPN13, and PTPN14.

Another aspect of the invention is a method of screening test substancesfor use as anti-cancer agents. A test substance is contacted with awild-type form of a protein tyrosine phosphatase or a mutant form of aprotein tyrosine phosphatase which is mutated in cancer cells. Activityof the form of the protein tyrosine phosphatase is tested. A testsubstance which increases the activity of the form of a protein tyrosinephosphatase is a potential anti-cancer agent. The protein tyrosinephosphatase is selected from the group consisting of: PTPRF, PTPRG,PTPRT, PTPN3, PTPN13, and PTPN14.

One embodiment of the invention provides an isolated, mutant form of aprotein tyrosine phosphatase. The phosphatase is selected from the groupconsisting of: PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14. Enzymaticactivity of the mutant form is reduced compared to wild-type.

Another embodiment of the invention provides an isolated polynucleotidewhich encodes a mutant form of protein tyrosine phosphatase. Thephosphatase is selected from the group consisting of: PTPRF, PTPRG,PTPRT, PTPN3, PTPN13, and PTPN14. Enzymatic activity of the mutant formis reduced compared to wild-type.

Still another aspect of the invention is a method of categorizingcancers. The coding sequence for or the amino acid sequence of one ormore protein tyrosine phosphatase family members in a sample of a cancertissue is determined The family member is selected from the groupconsisting of PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14.

A somatic mutation of the one or more protein tyrosine phosphatasefamily members is identified in the cancer tissue. The cancer tissue isassigned to a group based on the presence or absence of the somaticmutation.

According to another aspect of the invention a method of inhibitinggrowth of cancer cells is provided. A polynucleotide encoding awild-type protein tyrosine phosphatase is administered to cancer cells.The phosphatase is selected from the group consisting of PTPRF, PTPRG,PTPRT, PTPN3, PTPN13, and PTPN14, Growth of the cancer cells is therebyinhibited.

Yet another aspect of the invention is a method of identifying cancercells in a sample collected from a human. The coding sequence for or theamino acid sequence of one or more protein tyrosine phosphatase familymembers in a sample collected from the human is determined The familymember is selected from the group consisting of PTPRF, PTPRG, PTPRT,PTPN3, PTPN13, and PTPN14. The sample is selected from the groupconsisting of a suspected cancer tissue, blood, serum, plasma, andstool. A somatic mutation of said one or more protein tyrosinephosphatase family members is identified in the cancer tissue. Thesample is identified as containing cancer cells if a somatic mutation isidentified.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with reagents andmethods for detection, diagnosis, therapy, and drug screening pertainingto cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Distribution of mutations in PTPRT, PTPN13, PTPN14, PTPRG,PTPRF, and PTPN3. Black arrows indicate location of missense mutations,red arrows indicate location of nonsense mutations or frameshifts, andboxes represent functional domains (B41, band 41; CA, carbonicanhydrase; FN3, fibronectin type III; IG, immunoglobulin; MAM,meprin/A5/PTPμ; PDZ, postsynaptic density, discs large, zonulaoccludans; PTPc, catalytic phosphatase domain). Black stars indicatePTPRT mutants evaluated for phosphatase activity (see results in FIG.2), and red star indicates PTPRT mutant evaluated for effects on cellproliferation (see results in FIG. 3).

FIG. 2A-2B. Evaluation of phosphatase activity of mutant PTPRT. (FIG.2A) Saturation kinetics of wild-type and mutant PTPRT. His-taggedversions of PTPRT protein segments comprising the two catalytic domainscontaining wild-type (WT) and tumor-specific mutant sequences wereexpressed in bacteria and purified using nickel affinity chromatography.Equal amounts of WT and mutant proteins were used to evaluate enzymekinetics. The rate of hydrolysis of substrate (DiFMUP) is plottedagainst increasing substrate concentration. Data were fitted to theMichaelis-Menton equation and the resulting kinetic parameters of WT andmutant proteins are indicated (FIG. 2B).

FIG. 3A-3B. PTPRT overexpression suppresses growth of human cancercells. (FIG. 3A) HCT116 colorectal cancer cells were transfected withwild-type (WT) PTPRT construct, truncated R632X mutant PTPRT construct,or empty pCI-Neo vector. The photographs show colonies stained withcrystal violet after 14 days of geneticin selection. (FIG. 3B) Number ofresistant colonies (mean of two 25 cm² flasks) for WT PTPRT, mutantPTPRT, and empty vector.

FIG. 4. Expression analysis of PTPRT. PTPRT expression was evaluated byreverse transcription of total RNA followed by PCR amplification using aforward primer from exon 31 and a reverse primer from exon 32.Expression analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was performed as a control. Tissues analyzed are indicated above. PBLrepresents peripheral blood leukocytes; DiFi and Hct116 represent cancercell lines derived from the colon.

FIG. 5. (Table S1.) Mutations of the tyrosine phosphatome in humancancers

FIG. 6. (Table S2.) Tyrosine phosphatome genes analyzed (SEQ ID NO:6330-6416; encoded amino acids SEQ ID NO: 6417-6833).

FIG. 7. (Table S3.) Primers used for PCR amplification (forward SEQ IDNOs: 1 -2105 ; reverse SEQ ID NOs: 2106- 4210) and sequencing (SEQ IDNOs: 4211-6315)

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that protein tyrosine phosphatase genesare the targets of somatic mutations in cancers, suggesting that thesegenes function as tumor suppressors in human cells. 87 genes wereidentified as being members of the protein tyrosine phosphatasesuperfamily. These include members of the (i) the classical PTPs, (ii)the dual specificity phosphatases (DSPs), and (iii) the low molecularweight phosphatases (LMPs). See FIG. 6. Screening a collection ofcolorectal cancers identified six different genes of the classical PTPswhich are the object of somatic mutations in the colorectal cancers.Some of these genes are also the subject of somatic mutations in breast,lung, and/or gastric cancers. Screening collections of other types ofcancers will undoubtedly uncover other sets of the superfamily which aresomatically mutated. Other types of cancers which can be screened formutations include: bladder, melanoma, breast, non-Hodgkin's lymphoma,pancreatic, endometrial, prostate, kidney, skin, leukemia, thyroid, andlung.

Phosphatases which can be screened can be chosen from those shown inFIG. 6. Other phosphatases may be identified for screening, for example,using different bioinformatics criteria as described below. The primersidentified below for amplification and sequencing of the phosphatasescan be used, or other primers can be used as is convenient for thepractitioner. For identification of mutations, determined sequences insamples can be compared to known sequences in the literature. Forexample, for each of the 87 phosphatases of FIG. 6, a GenBank and aCelera accession number are provided. Sequences determined in samplescan be compared to the wild-type sequences provided in the databases andattached sequence listing. However, a better indication of involvementin cancer is provided by comparing a determined sequence to that in anormal tissue of the same human. Such a comparison indicates that achange is a somatic mutation. Sequences in databases refer to thesequences that existed in the databases as of May 14, 2004. GenBankstores and records sequences according to the dates on which they wereindicated as the most recent update. Thus the sequences available on May14, 2004 are maintained and publicly available. See also the sequencelisting. It is well recognized in the art that there is variation in thehuman population of wild-type protein and nucleic acid sequences. Suchvariation typically maintains sequences within a 95% identity range,more typically within a 97% identity range, and more typically within a99% identity range.

Matched pairs of cells for determining somatic mutations ideally arecells from a single individual. Typically the cells are of the sametype, e.g., lung cancer cells and normal lung cells. If a body samplesuch as blood or stool is being examined, then normal cells can beselected from any body tissue as a comparator.

Mutations that are relevant to cancer can be in almost any region of thephosphatases, because the relevant mutations are loss-of-functionmutations. Thus the mutations can be in the catalytic domain or in otherportions of the protein. Mutations can also be in non-coding, regulatoryregions of the gene. Non-synonymous mutations change the encoded aminoacids of a protein. Thus such mutations are highly likely to befunctionally relevant to cancer. Mutations in residues that areevolutionarily conserved among species are also highly likely to befunctionally relevant to cancer.

Since loss of protein tyrosine phosphatase activity appears to bedetrimental to cells, reacquisition of activity should have a positive,therapeutic effect. Test substances can be tested for their ability toenhance the activity of PTPs by contacting a wild-type or mutant PTPwith a test substance. The PTP can be isolated from cells and contactedin a cell-free system, or the PTP may be in cells, either geneticallyengineered host cells or native cells which express the PTP. The cellscan be tested in culture or in a model non-human animal system.Typically the cells will be somatic cells. The PTP can be any mutant orwild-type form, especially one of the six PTPs identified as mutant incolorectal cancers, but also may any of the 87 identified below. One ofthe mutant forms identified in the present study can be used (see FIG.5) or other mutant forms, for example those found in other types ofcancer. PTPs can be isolated from producing host cells or nativeproducer cells. One means of purifying a PTP is disclosed in example 7,in which a His tag is added to the PTP by cloning and then used topurify the PTP using nickel affinity beads. A desirable test substancefor becoming a candidate anti-cancer agent will enhance the activity ofa wild-type and/or mutant PTP or enhance PTP activity of a cell withwild-type, mutant, or both types of PTP.

Polynucleotides comprising coding sequences for PTPs, in particularmutant PTPs found in cancer cells, can be naturally occurring codingsequences or coding sequences which are synthesized based on the geneticcode and the amino acid sequence of a mutant PTP. The coding sequencescan be inserted in expression vectors so that quantities of the mutantPTPs can be produced efficiently and used in drug screening assays.Alternatively, host cells which contain expression vectors encodingmutant PTPs can be used for drug screening assays. The mutant PTPs maybe reduced in enzyme activity, for example with a higher K_(m) or with alower K_(cat) than wild-type. The mutant PTPs may alternatively have nodetectable enzymatic activity.

Isolated polynucleotides are polynucleotides which are separated fromthe chromosome upon which they normally reside in the human genome. Theyare typically separated from the genes which flank them on a normalhuman chromosome. They may be in a vector with an origin of replication,or they may simply be an isolated linear piece of nucleic acid. Thepolynucleotides encoding PTPs may or may not contain the introns whichare present in the human genome.

Cancer tissues can be categorized on the basis of which, if any,phosphatase mutation(s) they contain. Any of the PTPs demonstrated toharbor cancer-related mutations can be used for the categorization.Somatic mutations are identified on the basis of a difference between anaffected tissue and a normal tissue of the same individual.Categorization of the tissue can be used for stratifying patients forclinical trials, for analyzing data from clinical trials, forcorrelating with prognostic data (such as recurrence, metastasis, andlife expectancy), as well as for selecting an appropriate course oftreatment for a cancer patient. The PTP categorization can be used inconjunction with other data, for example, histopathological data, toidentify a cancer. Similarly, PTP somatic mutation analysis can be usedin any tissue or body sample to diagnose cancer. Presence of a mutantPTP or coding sequence in a tissue or body sample indicates the presenceof cancer cells, either in the sample itself, or in a tissue whichdrains into the sample. Thus, for example, detection of PTP mutants in afecal sample reflects the presence of colorectal cancer cells in thehuman from whom the sample was taken. Body samples which can be testedinclude without limitation suspected cancerous tissues, stool, sputum,tears, saliva, blood, plasma, serum, urine, and bronchoalveolar lavage.

The mutational data associating loss of PTP function with cancersstrongly suggests that PTPs are tumor suppressors. Therefore wild-typePTP coding sequences can be used as therapeutic agents for treatingtumors. Wild-type PTP coding sequences are shown in the sequencelisting. These sequences or wild-type sequences which are at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical, or least 99% identical, can be used to deliver wild-type PTPto tumor cells. The coding sequences may or may not contain introns.Sequences for any of the six PTPs identified as somatically mutated incolorectal cancers may be used, as well as any of the other PTPsidentified in FIG. 6.

Viral or non-viral vectors may be used for delivery of polynucleotides.For example, adenoviruses, adeno-associated viruses, herpes viruses, andretroviruses can be used for delivery. Non-viral vectors includeliposomes, nanoparticles and other polymeric particles. Any vectors ortechniques known in the art may be used for delivering genes to cells orhumans. See, e.g., Gene Therapy Protocols, Paul D. Robbins, ed., HumanPress, Totowa, N.J., 1997. Vectors may not be necessary according tosome protocols, and coding sequences can be administered without a meansof replication. Administration of gene therapy vectors can be by anymeans known in the art, including but not limited to intravenous,intramuscular, intratumoral, intranasal, intrabronchial, andsubcutaneous injections or administration. An effective amount ofpolynucleotide is one which inhibits growth of cancer cells in ameasurable amount. Preferably the tumor regresses and shrinks, or atleast ceases to grow larger.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Identification of PTP Gene Superfamily Members

We employed a combination of Hidden Markov Models representing catalyticdomains of members of the PTP superfamily to identify 53 classical PTPs(21 RPTPs and 32 NRPTPs), 33 DSPs, and one LMP in the human genome (12).This analysis revealed a set of genes representing all known human PTPs(13) as well as seven putative PTPs.

EXAMPLE 2 Identification of PTP Gene Superfamily Members with Mutations

As an initial screen to evaluate whether these phosphatases aregenetically altered in human cancer, we analyzed the coding exons of all87 members of this gene family in 18 colorectal cancers. A total of 1375exons from all annotated RPTPs, NRPTPs, DSPs and LMPs were extractedfrom genomic databases (12). These exons were PCR-amplified from cancergenomic DNA samples and directly sequenced using dye terminatorchemistry (12). Whenever a presumptive mutation was identified, weattempted to determine whether it was somatically acquired (i.e., tumorspecific) by examining the sequence of the gene in genomic DNA fromnormal tissue of the relevant patient.

From the 3.3 Mb of sequence information obtained, we identified sixgenes containing somatic mutations, including three members of the RPTPsubfamily (PTPRF, PTPRG, and PTPRT) and three members of the NRPTPsubfamily (PTPN3, PTPN13 and PTPN14). These six genes were then furtheranalyzed for mutations in another 157 colorectal cancers. Through thisstrategy we identified 77 mutations in the six genes, in aggregateaffecting 26% of the colorectal tumors analyzed (Table S1, FIG. 1).Examination of these six genes in seven other tumor types identifiedPTPRT mutations in two of 11 (18%) lung cancers and two of 12 gastriccancers (17%), and PTPRF mutations in one of 11 (9%) lung cancers andone of 11 (9%) breast cancers. No mutations were identified in 12pancreatic cancers, 12 ovarian cancers, 12 medulloblastomas or 12glioblastomas (FIG. 5 (Table S1), FIG. 1). In total, 83 nonsynonymousmutations were observed, all of which were somatic in the cancers thatcould be assessed (12).

Fifteen of the 83 mutations were nonsense, frameshift or splice sitealterations, all of which were predicted to result in aberrant ortruncated proteins. In 16 tumors both alleles of the phosphatase geneappeared to be mutated, a characteristic often associated with tumorsuppressor genes. The majority of tumors with PTP gene mutations alsocontained mutations in KRAS or BRAF, and nine tumors containedalterations in previously reported tyrosine kinase genes (FIG. 5 (TableS1)). Thus the mutant phosphatases identified in this study are likelyto operate through cellular pathways distinct from those associated withpreviously identified mutant kinases.

EXAMPLE 3 Analysis of Mutation Types

Analysis of mutations in tumors is complicated by the fact thatmutations can arise either as functional alterations affecting key genesunderlying the neoplastic process or as non-functional “passenger”changes. The multiple waves of clonal expansion and selection that occurthroughout tumorigenesis lead to fixation of any mutation that hadpreviously occurred in any predecessor cell, regardless of whether themutation was actually responsible for the clonal expansion. Twoindependent lines of evidence suggest that the sequence alterations weobserved are functional. First, the ratio of nonsynonymous to synonymousmutations provides an indication of selection, as synonymous alterationsusually do not exert a growth advantage. There were no somaticsynonymous mutations detected in the colorectal cancers analyzed,resulting in a ratio of nonsynonymous to synonymous mutations of 77 to0, much higher than the expected 2:1 ratio for non-selected passengermutations (p<1×10−6). Second, the prevalence of mutations in the codingregions of the analyzed genes was ˜19 per Mb of tumor DNA, similar tothe prevalence of functional somatic alterations observed in other genefamilies (e.g., the tyrosine kinome (6)) and significantly higher thanthe prevalence of nonfunctional alterations previously observed in thecancer genomes (˜1 per Mb, p<0.01)(14). These data support the idea thatthese mutations were the targets of selection during tumorigenesis.

EXAMPLE 4 Effect of Point Mutations on Enzymatic Activity

The great majority of the nonsense and frameshift mutations (FIG. 1)would result in polypeptides devoid of C-terminal phosphatase catalyticdomains, thereby leading to inactivation of the phosphatase. To evaluatewhether tumor-specific point mutations alter phosphatase activity, webiochemically tested mutant versions of PTPRT, the most frequentlymutated PTP in the superfamily. Mutations in both intracellular PTPdomains (D1 and D2) were evaluated. His-tagged versions of the catalyticregion of wild-type PTPRT, two D1 mutants (Q987K and N11281), and threeD2 mutants (R1212W, R1346L and T1368M) were produced in bacteria andanalyzed for phosphatase activity using6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate(FIG. 2) (12). All D1 and D2 mutants had reduced phosphatase activitycompared with the wild-type protein (FIG. 2). Interestingly, the kineticparameter K_(cat) was reduced in both D1 mutants, while K_(m) wasincreased in all three D2 mutants, suggesting that mutations in the twodomains may have different effects on enzymatic activity. Although theD2 domain has been thought to usually be catalytically inactive (1),these results are consistent with recent studies that show that the D2domain is important for phosphatase activity in some receptorphosphatases (15). These biochemical data on missense mutations, coupledwith the large number of truncating mutations noted above, suggest thatPTPRT functions as a tumor suppressor gene.

EXAMPLE 5 Phosphatase Functions as Tumor Suppressor

To determine whether PTPRT inhibits tumor cell growth, we transfectedwild-type PTPRT into HCT116 colorectal cancer cells (12). An identicalexpression vector containing an R632X mutant of PTPRT was used forcomparison. Wild-type PTPRT potently inhibited cell growth in thisassay, as seen by the substantial decrease in the number of neomycinresistant colonies compared with the R632X mutant or with vector alone(FIGS. 3A, 3B). Similar results with wild-type and mutant PTPRT werealso observed in DLD1 colorectal cancer cells.

EXAMPLE 6 Discussion

The combination of these genetic, biochemical, and cellular data suggestthat PTPRT and the other identified phosphatases are likely to act astumor suppressors. This is consistent with the function of otherphosphatases implicated in tumorigenesis (7,8,16), and with the generalrole of phosphatases in inhibiting various growth promoting signalingpathways (2). The absence of biallelic mutations in a subset of theanalyzed tumors suggests that some alterations may act in a dominantnegative fashion or may affect gene dosage, mechanisms that have beenpreviously involved in inactivation of other tumor suppressor genes (17,18).

Little is known about the functional role of the tyrosine phosphatasesdiscussed here. PTPN13 appears to be involved in apoptosis (19) and maybe partly responsible for the anti-tumor effects of tamoxifen (20).Overexpression of PTPN3 inhibits growth of NIH/3T3 cells, possiblythrough interaction with valosin containing protein (VCP/p97) (21).PTPN14 and PTPRF are thought to play a role in cell adhesion byregulating tyrosine phosphorylation of adherens junction proteins (22,23). As increased phosphorylation of adherens junctions has been shownto increase cell motility and migration (22, 24), mutationalinactivation of these genes may be an important step in cancer cellinvasion and metastasis. PTPRG maps to chromosome 3p14.2, a regionfrequently lost in lung, renal and early stage breast tumors, and isthought to be a target of the translocation at 3p14 in familial renalcell carcinoma (25-27). However, no point mutations in PTPRG (28) or anyof the other genes identified here have been previously described in anycancer. PTPRT is expressed in the developing central nervous system andin the adult cerebellum (29) and had not been thought to play a role inthe growth or differentiation of other tissues. We have found that PTPRTis expressed in a variety of human tissues, including normal colonepithelium as well as cells derived from colorectal cancers (FIG. 4).

EXAMPLE 7 Materials and Methods

Identification of PTP genes. Protein tyrosine phosphatase genes wereidentified by analysis of InterPro (IPR) phosphatase domains presentwithin the Celera draft human genome sequence. IPR003595, IPR000340,IPR000751 and IPRO02115 were used to search all known and predictedgenes for classical PTPs (RPTPs and NRPTPs), DSPs, DSPs related toCDC25, and LMPs, respectively. This resulted in identification of 91tyrosine phosphatases, three of which were pseudogenes and therefore notanalyzed further. PTEN, which has been determined to act primarily as alipid phosphatase was also not analyzed.

PCR, sequencing, and mutational analysis. Sequences for all availableannotated exons and adjacent intronic sequences of identified PTP, DSPand LMP genes were extracted from Celera draft human genome sequence(website: celera.com) or from GenBank (website: genbank.nlm.nih.gov).Celera and public accession numbers of all analyzed genes are availablein FIG. 6 (Table S2).

Primers for PCR amplification and sequencing were designed using thePrimer 3 program (website:genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi), and were synthesizedby MWG (High Point, N.C.) or IDT (Coralville, Iowa). PCR amplificationand sequencing were performed on tumor DNA from 18 early passage celllines as previously described (6) using a 384 capillary automatedsequencing apparatus (Spectrumedix, State College, Pa.). Sequence traceswere assembled and analyzed to identify potential genomic alterationsusing the Mutation Explorer software package (SoftGenetics, StateCollege, Pa.). Of the 1375 exons extracted, 92% were successfullyanalyzed, each in an average of 17 tumor samples. All mutations listedin Table 51 were determined to be somatic except in 10 cases in which nonormal tissue was available for comparison. Sequences of all primersused for PCR amplification and sequencing are available in FIG. 7 (TableS3).

Construction of wild-type and mutant PTPRT proteins. The region encodingtwo catalytic domains of PTPRT was cloned by PCR using PlatinumHi-fidelity Taq polymerase (Invitrogen, Carlsbad, Calif.) from humanfetal brain cDNA with primers GGAATTCCATATGGCCTTACCAGAGGGGCAGACAG (SEQID NO: 6316) and CGGGATCCCCCAGTTACTGCCATTCACA (SEQ ID NO: 6317) andcloned in frame fused to the 6×His tag of pET19b expression vector(Novagen, Madison, Wis.). The Q987K, N11281, R1212W, R1346L and T1368Mmutants were made using sexual PCR (Ref 30). The primersCAAAAGTCCTTTACAGTCTCCTTCATCGGACCTTGAGTCGCAATG (SEQ ID NO: 6318) andAAGGAGACTGTAAAGGACTTTTGGAG (SEQ ID NO: 6319) were used as mutagenicprimers for the Q987K mutant; the primersCCATGCTTGACATGGCCGAGATTGAAGGGGTGGTGGACATCTTC (SEQ ID NO: 6320) andATCTCGGCCATGTCAAGCATGG (SEQ ID NO: 6321) were used as mutagenic primersfor the N11281 mutant; the primersCAATGCTGCAGTCCTCGGGCCACACACGGGGTGTCACAATG (SEQ ID NO: 6322) andTGGCCCGAGGACTGCAGCATTG (SEQ ID NO: 6323) were used as mutagenic primersfor the R1212W mutant; the primersCTATACGATAACCATCCTGTGGCAGGGCCATGTTACAGATGCG (SEQ ID NO: 6324) andTGCCACAGGATGGTTATCGTATAG (SEQ ID NO: 6325) were used as mutagenicprimers for the R1346L mutant; and the primersGAGCGCTTGGAGGGGGGCATGTCCCGGTAGGCAGGCC (SEQ ID NO: 6326) andTGCCCCCCTCCAAGCGCTC (SEQ ID NO: 6327) were used as mutagenic primers forthe T1368M mutant. For expression of recombinant proteins, BL21-DE3bacteria were grown to late log phase and induced with 1 mM IPTG for 3hours at 37° C. Bacterial lysates were made by sonication in lysisbuffer (1 mM Tris, 1 M NaCl, 10 mM imidazole 0.1% igepal, pH 8.0) andincubated with Ni-NTA beads for 45 min at 4° C. The Ni-NTA beads werewashed with 50 mM imidazole buffer (40 mM Tris, 100 mM NaCl, 50 mMimidazole, pH8.0) and bound protein was eluted with 500 mM imidazole.

Phosphatase kinetic analysis. Various concentrations of6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP—Molecular ProbesD6567) were incubated with 800 ng of purified protein in 40 mM Tris-HClpH8.0, 100 mM NaCl, 5 mM CaCl2, and 10 mM DTT in a 100 uL reaction. Thereaction was incubated at 37° C. for 30 minutes and fluorometricmeasurements were taken at an excitation wavelength of 360 nm and anemission wavelength of 460 nm and extrapolated to a standard curve of6,8-difluoro-4-methylumbelliferone. The data were fitted to theMichaelis-Menton equation using GraphPad Prism v. 3.02.

Cell proliferation assays. Full length wild-type or R632X mutant PTPRTcDNA sequences were cloned into the pCI-Neo vector (Promega, Madison,Wis.). Subconfluent HCT116 and DLD1 colorectal cancer cells weretransfected with equal amounts of the wild-type PTPRT construct, R632Xmutant PTPRT construct, or empty vector and grown for 48 hours. Cellswere then trypsinized and plated in T25 flasks with fresh mediacontaining geneticin. Cells were grown for 2-3 weeks and stained withcrystal violet. The expression level and the mutational status of PTPRTare not known in either HCT116 or DLD1 cells as no normal tissues fromthe same patients are available as controls.

Expression analysis. Total RNAs from various human tissues werepurchased form BD Bioscience (San Jose, Calif.) and equal amounts werereverse transcribed into cDNAs with random primers. PTPRT expression wasexamined by PCR using primers CCACATCGTGAAAACACTGC (SEQ ID NO: 6328) andCAACAGGAGACCCCTCAGAA (SEQ ID NO: 6329) which are located in exons 31 and32 and result in a 284 by product.

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

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1. A method for identifying mutations involved in cancer, comprising:determining nucleotide sequence differences in a human nucleotidesequence in matched pairs of cancer cells and normal cells, each pairbeing from a single individual, wherein the human nucleotide sequenceencodes a protein tyrosine phosphatase selected from the groupconsisting of: PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14.
 2. Themethod of claim 1 wherein at least one of the nucleotide sequencedifferences is in a region encoding a catalytic domain.
 3. The method ofclaim 1 wherein at least one of the nucleotide sequence differences is apoint mutation.
 4. The method of claim 1 wherein at least one of thenucleotide sequence differences is in a regulatory region.
 5. The methodof claim 1 further comprising determining whether a nucleotide sequencedifference is synonymous or non-synonymous.
 6. The method of claim 1further comprising determining whether a nucleotide difference affectsan evolutionarily conserved amino acid residue.
 7. The method of claim 1wherein the protein tyrosine phosphatase is PTPRT.
 8. The method ofclaim 1 wherein the protein tyrosine phosphatase is PTPRG.
 9. The methodof claim 1 wherein the cancer cells are selected from the groupconsisting of bladder cancer, melanoma, breast cancer, non-Hodgkin'slymphoma, colon and rectal cancer, pancreatic cancer, endometrialcancer, prostate cancer, kidney cancer (renal cell), skin(non-melanoma), leukemia, thyroid cancer, and lung cancer.
 10. A methodof screening test substances for use as anti-cancer agents, comprising:contacting a test substance with a wild-type form or a mutant form of aprotein tyrosine phosphatase which is mutated in cancer cells, whereinthe protein tyrosine phosphatase is selected from the group consistingof: PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14; testing activity ofthe form of the protein tyrosine phosphatase, wherein a test substancewhich increases the activity of the form of a protein tyrosinephosphatase is a potential anti-cancer agent.
 11. The method of claim 10wherein the form of a protein tyrosine phosphatase is in a cell.
 12. Themethod of claim 10 wherein the form of a protein tyrosine phosphatase isisolated from a cell.
 13. The method of claim 10 wherein the form of aprotein tyrosine phosphatase is in a cell of a cancer cell line.
 14. Themethod of claim 10 wherein the form of a protein tyrosine phosphatase isin a cell which has been modified to express the form of a proteintyrosine phosphatase.
 15. The method of claim 10 wherein the form of aprotein tyrosine phosphatase is PTPRF.
 16. The method of claim 10wherein the form of a protein tyrosine phosphatase is PTPRG.
 17. Themethod of claim 10 wherein the form of a protein tyrosine phosphatase isPTPRT.
 18. The method of claim 10 wherein the form of a protein tyrosinephosphatase is PTPN3.
 19. The method of claim 10 wherein the form of aprotein tyrosine phosphatase is wild-type.
 20. The method of claim 10wherein the form of a protein tyrosine phosphatase is PTPN13.
 21. Themethod of claim 10 wherein the form of a protein tyrosine phosphatase isPTPN14.
 22. The method of claim 15 wherein the PTPRF has a mutationselected from the group consisting of R218C, G645R, G1040V, R1333C,V1390I, 387-468 substitution, and A381V.
 23. The method of claim 16wherein the PTPRG has a mutation selected from the group consisting ofT361M, A462V, T514M, R593W, E955G, Y973C, R1312W, and I1326V.
 24. Themethod of claim 17 wherein the PTPRT has a mutation selected from thegroup consisting of A209T, V1269M, A209T, F248S, Y280H, Y412F, N510K,T605M, V648G, R632X, A707T, D927G, A707V , R1021X, F74S, L708P, R975X ,LOH, Q987K, A1118P, T1368M, N1128I, R1212W, M1259L, I395V, Y1351F,T1368M, R453C, K218T, R1346L, and R790I.
 25. The method of claim 18wherein the PTPN3 has a mutation selected from the group consisting ofV154I, A239V, E610X, S300A, F307L, and R330Q.
 26. The method of claim 20wherein the PTPN13 has a mutation selected from the group consisting ofH2Q, E952X, G1476C, R380X, M2443I, R402X, S443N, A529D, Q1691X, K2131N,D2154H, R2205W, R2338X, Y2279X, M2307T, I2458V, and E2474D.
 27. Themethod of claim 21 wherein the PTPN14 has a mutation selected from thegroup consisting of L56M, R293Q, S314P, Q332R, R491Q, P525L, H633Y,P528L, T657M, E883D, and T1068M.
 28. An isolated, mutant form of proteintyrosine phosphatase selected from the group consisting of: PTPRF,PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14, wherein enzymatic activity ofthe mutant form is reduced compared to wild-type.
 29. The isolated,mutant form of claim 28 which is PTPRF.
 30. The isolated, mutant form ofclaim 28 which is PTPRG.
 31. The isolated, mutant form of claim 28 whichis PTPRT.
 32. The isolated, mutant form of claim 28 which is PTPN3. 33.The isolated, mutant form of claim 28 which is PTPN13.
 34. The isolated,mutant form of claim 28 which is PTPN14.
 35. The isolated, mutant formof claim 29 wherein the PTPRF has a mutation selected from the groupconsisting of R218C, G645R, G1040V, R1333C, V1390I, 387-468substitution, and A381V.
 36. The isolated, mutant form of claim 30wherein the PTPRG has a mutation selected from the group consisting ofT361M, A462V, T514M, R593W, E955G, Y973C, R1312W, and I1326V.
 37. Theisolated, mutant form of claim 31 wherein the PTPRT has a mutationselected from the group consisting of A209T, V1269M, A209T, F248S,Y280H, Y412F, N510K, T605M, V648G, R632X, A707T, D927G, A707V, R1021X,F74S, L708P, R975X, LOH, Q987K, A1118P, T1368M, N1128I, R1212W, M1259L,I395V, Y1351F, T1368M, R453C, K218T, R1346L, and R790I.
 38. Theisolated, mutant form of claim 32 wherein the PTPN3 has a mutationselected from the group consisting of V154I, A239V, E610X, S300A, F307L,and R330Q.
 39. The isolated, mutant form of claim 33 wherein the mutantPTPN13 has a mutation selected from the group consisting of H2Q, E952X,G1476C, R380X, M2443I, R402X, S443N, A529D, Q1691X, K2131N, D2154H,R2205W, R2338X, Y2279X, M2307T, I2458V, and E2474D.
 40. The isolated,mutant form of claim 34 wherein the mutant PTPN14 has a mutationselected from the group consisting of L56M, R293Q, S314P, Q332R, R491Q,P525L, H633Y, P528L, T657M, E883D, and T1068M.
 41. An isolatedpolynucleotide which encodes a mutant form of protein tyrosinephosphatase selected from the group consisting of: PTPRF, PTPRG, PTPRT,PTPN3, PTPN13, and PTPN14, wherein enzymatic activity of the mutant formis reduced compared to wild-type.
 42. A host cell comprising thepolynucleotide of claim
 41. 43. A method of categorizing cancers,comprising: determining the coding sequence for or the amino acidsequence of one or more protein tyrosine phosphatase family membersselected from the group consisting of PTPRF, PTPRG, PTPRT, PTPN3,PTPN13, and PTPN14 in a sample of a cancer tissue; identifying a somaticmutation of said one or more protein tyrosine phosphatase family membersin the cancer tissue; assigning the cancer tissue to a group based onthe presence or absence of the somatic mutation.
 44. The method of claim43 wherein the mutation is one which truncates the protein tyrosinephosphatase family member.
 45. The method of claim 43 wherein themutation is non-synonymous.
 46. The method of claim 43 wherein themutation is somatic.
 47. The method of claim 43 wherein the mutation isa point mutation.
 48. The method of claim 43 wherein the protein kinasefamily member is PTPRF.
 49. The method of claim 43 wherein the proteinkinase family member is PTPRG.
 50. The method of claim 43 wherein theprotein kinase family member is PTPRT.
 51. The method of claim 43wherein the protein kinase family member is PTPN3.
 52. The method ofclaim 43 wherein the protein kinase family member is PTPN13.
 53. Themethod of claim 43 wherein the protein kinase family member is PTPN14.54. The method of claim 43 wherein the group is used to analyze ordesign clinical trials.
 55. The method of claim 43 wherein the group isused to correlate with prognostic data.
 56. The method of claim 43wherein the group is used to correlate with recurrence data.
 57. Themethod of claim 43 wherein the group is used to select an appropriatetherapeutic agent.
 58. The method of claim 43 wherein the group is usedto identify cancer.
 59. The method of claim 43 wherein the cancer tissueis colorectal.
 60. The method of claim 43 wherein the cancer tissue isbreast.
 61. The method of claim 43 wherein the cancer tissue is lung.62. The method of claim 43 wherein the cancer tissue is gastric.
 63. Amethod of inhibiting growth of cancer cells, comprising: administeringto cancer cells a polynucleotide encoding a wild-type protein tyrosinephosphatase selected from the group consisting of PTPRF, PTPRG, PTPRT,PTPN3, PTPN13, and PTPN14, whereby growth of the cancer cells isinhibited.
 64. The method of claim 63 wherein the cancer cells arecolorectal.
 65. The method of claim 63 wherein the cancer cells arebreast.
 66. The method of claim 63 wherein the cancer cells are lung.67. The method of claim 63 wherein the cancer cells are gastric.
 68. Themethod of claim 63 wherein the cancer cells are in culture.
 69. Themethod of claim 63 wherein the cancer cells are in a human body.
 70. Themethod of claim 63 wherein the polynucleotide is administered byintratumoral injection.
 71. The method of claim 63 wherein the cancercells comprise one or two mutant alleles of a protein tyrosinephosphatase selected from the group consisting of PTPRF, PTPRG, PTPRT,PTPN3, PTPN13, and PTPN14.
 72. A method of identifying cancer cells in asample collected from a human, comprising: determining the codingsequence for or the amino acid sequence of one or more protein tyrosinephosphatase family members selected from the group consisting of PTPRF,PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14 in a sample collected from thehuman, wherein the sample is selected from the group consisting of asuspected cancer tissue, blood, serum, plasma, and stool; identifying asomatic mutation of said one or more protein tyrosine phosphatase familymembers in the cancer tissue; identifying the sample as containingcancer cells if a somatic mutation is identified.
 73. The method ofclaim 72 wherein the protein tyrosine phosphatase family member isPTPRF.
 74. The method of claim 72 wherein the protein tyrosinephosphatase family member is PTPRG.
 75. The method of claim 72 whereinthe protein tyrosine phosphatase family member is PTPRT.
 76. The methodof claim 72 wherein the protein tyrosine phosphatase family member isPTPN3.
 77. The method of claim 72 wherein the protein tyrosinephosphatase family member is PTPN13.
 78. The method of claim 72 whereinthe protein tyrosine phosphatase family member is PTPN14.
 79. The methodof claim 1 wherein the protein tyrosine phosphatase is PTPRF.
 80. Themethod of claim 1 wherein the protein tyrosine phosphatase is PTPN3. 81.The method of claim 1 wherein the protein tyrosine phosphatase is PTPN1382. The method of claim 1 wherein the protein tyrosine phosphatase isPTPN14.